Coenzyme B12 (aka, adenosylcobalamin, AdoCbl) is synthesized only by prokaryotes, and is essential to the survival of many pathogens. Hence, precise knowledge of the biochemistry underpinning this major pathway (~25 reactions), and an understanding of the structural properties of the enzymes involved, is critical to the design of inhibitors that could be used to target disease-causing bacteria or any other prokaryote of societal importance. I will apply a multidisciplinary approach to study the last step of AdoCbl biosynthesis, which is catalyzed by the AdoCbl-P phosphatase (CobC) enzyme. There are two parts to my proposal: i) Functional and topological studies of CobC; ii) analysis of interactions of CobC with other proteins suspected to comprise a multiprotein complex localized to the cell membrane. I will collaborate with structural biologists led by Ivan Rayment (Department of Biochemistry, UW-Madison) to gain insights into the mechanism of function of CobC, and to advance our understanding of the implications of the existence of a membrane associated biosynthetic complex. PUBLIC HEALTH RELEVANCE: Coenzyme B12 is only synthesized by prokaryotes, including many pathogens, and it is essential to the survival of animals, including humans. A better understanding of the biochemistry of coenzyme B12 biosynthesis will facilitate the design of antimicrobials that could be used to target disease-causing bacteria. In collaboration with others, our laboratory used knowledge gained from the studies of coenzyme B12 synthesis in our fight against cancer. A multifaceted approach will be applied to learn more about the last step of the pathway, which, in Salmonella enterica, it may occur outside the cell.